With the exception of one other study that observed single gene bursting directly (Yunger, et al. (2010)), all other studies in mammalian cells are based on measuring protein distributions and inferring transcriptional activity.
To ascertain whether a causal relationship exists between activation of autophagy and metabolic stress in breast cancer cells, we measured protein synthesis activity and ATP level in MCF-7 cells subjected to nutrient starvation.
To measure protein translation activity, lymphoblastoid cells were first serum-starved for 8 h to synchronize cell growth before re-stimulated with a normal concentration of serum.
Such techniques are valuable to measure protein expression, solubility, activity, and native posttranslational modification events in heterologous systems.
In order to measure the pathway activity, we first measured Ras protein activity levels able to stimulate the ERK cascade.
An optimized peptide substrate was used to measure protein kinase B (PKB) activity in single cells.
Three μl were diluted 1 10 in water and the dilutions were used to measure protein concentration and chitinase activity (in 96-well plates).
First, HAECs were infected with rAd-GLO1 at a wide range of multiplicity of infection (MOI), from 1 to 50, and measured protein expression and enzymatic activity of GLO1 (Fig. S5A C).
Currently there are no methods to measure functional protein activity reliably.
In order to validate our hypothesis and this approach, we selected two heterologous genes of interest, applied the approach as outlined above, expressed the resulting optimized genes in S. cerevisiae, and measured resulting protein activity.
